Project Summary The proposed program responds to the objectives of RFA RM-15-008 (?Novel and innovative tools to facilitate identification, tracking, manipulation, and analysis of glycans and their functions?) by providing a reliable approach for the production of anti-glycan and anti-glycolipid monoclonal antibodies. Using a virus-like particle (VLP) platform displaying short synthetic glycans, we have elicited high affinity, highly mutated IgG B cell responses against microbial and mammalian sugars. Antibody production was entirely dependent on CD4 T cell help and the presence of an NKT cell adjuvant. While exquisitely specific for the target glycan, as tested using a glycan microarray, these antibodies exhibited nanomolar affinity, a remarkable and unusual feature for anti-glycan antibodies, associated to a large number of somatic mutations in both heavy and light chains. In a separate set of experiments, we also produced anti-glycolipid polyclonal antibodies that could discriminate neck and head group, as well as anomerization of the proximal sugar. To advance the field and make our approach widely usable and available, we will carry out two specific aims: Aim 1, Approaches to obtain nanomolar affinity anti-glycan antibodies and analytical methods to characterize anti-glycan responses, in which we will refine our approach of glycan synthesis, coupling to VLP, and production of antibodies by using glycan microarray, to generalize a principle that allows the production of highly specific nanomolar anti-glycan monoclonal antibodies. Aim 2, ?Development of approaches to obtain anti- glycolipid monoclonal antibodies of high affinity and specificity? in which the observation made in rabbits will be translated to mice in order to produce monoclonal antibodies specific for glycolipids and usable for the analysis of their fine chemical structure. In addition, a glycolipid microarray will be developed to facilitate antibody characterization. The benefits of our approaches to produce new tools to study glycans and glycolipids are obvious. Indeed, such antibodies will give access to a number of assays that were impossible or utterly challenging in the field of glycan and glycolipid analysis such as Western blotting, immunoprecipitation, in vivo targeting, direct chemical determination, to only cite a few.